Engineering of a GSH activatable photosensitizer for enhanced photodynamic therapy through disrupting redox homeostasis

Although disrupted redox homeostasis has emerged as a promising approach for tumor therapy, most existing photosensitizers are not able to simultaneously improve the reactive oxygen species level and reduce the glutathione (GSH) level. Therefore, designing photosensitizers that can achieve these two aspects of this goal is still urgent and challenging. In this work, an organic activatable near-infrared (NIR) photosensitizer, CyI-S-diCF3, is developed for GSH depletion-assisted enhanced photodynamic therapy. CyI-S-diCF3, composed of an iodinated heptamethine cyanine skeleton linked with a recognition unit of 3,5-bis(trifluoromethyl)benzenethiol, can specifically react with GSH by nucleophilic substitution, resulting in intracellular GSH depletion and redox imbalance. Moreover, the activated photosensitizer can produce abundant singlet oxygen (1O2) under NIR light irradiation, further heightening the cellular oxidative stress. By this unique nature, CyI-S-diCF3 exhibits excellent toxicity to cancer cells, followed by inducing earlier apoptosis. Thus, our study may propose a new strategy to design an activatable photosensitizer for breaking the redox homeostasis in tumor cells.


Introduction
Redox homeostasis, a dynamic balance of reactive oxygen species (ROS) generation and elimination, is a signicant index for cell metabolism. [1][2][3][4][5] ROS (e.g., anion superoxide, hydroxyl radical and singlet oxygen) are usually generated by intracellular oxidoreductase-catalyzed processes and exogenous light stimulation. [6][7][8][9] The elevated ROS could promote the proliferation of tumors, whereas an excessive amount of ROS would elicit cell death due to strengthened oxidative stress. 10,11 To equilibrate the high-level of oxidative stress, cancer cells would functionally enhance the endogenous antioxidant systems by producing glutathione (GSH), an important reducing agent enriched in tumor tissue. [12][13][14] Given this unique feature, breaking the cellular redox homeostasis to induce apoptosis would be recognized as a promising strategy for tumor therapy. Recently, numerous nanomaterials have been constructed to disrupt the redox homeostasis by boosting the ROS level and concurrently decreasing GSH concentrations, leading to cell apoptosis. [15][16][17][18][19] However, there are still very few organic photosensitizers (PSs) able to simultaneously achieve the amplication of ROS level and down-regulation of GSH content in tumor cells. 20 Thus, it is of great importance to rationally design and synthesis novel photosensitizers that exhibited excellent ROS generation ability and GSH depletion property, which, however, is still a great challenge.
Owing to its precise spatiotemporal selectivity, noninvasive and biosafety feature, photodynamic therapy (PDT) has regarded as a promising oncotherapy. [21][22][23][24][25][26] Photosensitizers are usually able to produce toxic singlet oxygen ( 1 O 2 ) to kill tumor cells through the energy transfer (type II) process between the triplet state in PSs and surrounding O 2 during photodynamic therapy. [27][28][29] However, though many photosensitizers have been used for PDT, the tumor suppression efficacy was still weak due to the limited photon utilization of the photosensitizers, which could be attributed to the short excitation wavelengths and low molar extinction coefficient. 30 Furthermore, on account of the aggressive proliferation of cancer cells, intracellular O 2 concentrations was lower than normal cells, which further restricted the 1 O 2 generation efficiency of photosensitizers in phototherapy. 31,32 To date, several studies have been reported that heptamethine cyanine dyes could be used as an effective photosensitizer for PDT as its near-infrared (NIR) excitation light and high molar extinction coefficient. Meanwhile, the 1 O 2 generation efficacy of these PSs could be distinctly improved by accelerating the intersystem crossing (ISC) process using heavyatom effect (HAE). [33][34][35][36][37] It is worth noting that the chloride atom at the meso position in heptamethine cyanine skeleton also could be easily modied by different groups to construct functional uorescence probes (e.g., GSH-activatable uorescence probe). [38][39][40] Therefore, heptamethine cyanine scaffold might provide an promising platform to design a novel photosensitizer with enhanced ROS production efficiency and high GSH consumption ability for breaking the redox homeostasis in tumor cells.
In this work, we developed a novel GSH-activated NIR photosensitizer (denoted as CyI-S-diCF 3 ) for GSH depletionassisted enhanced photodynamic therapy in tumor cells (Scheme 1). We rst employed iodinated heptamethine cyanine dye as the NIR photosensitizer scaffold because of its superior 1 O 2 generation capability and biocompatibility. To confer the GSH responsiveness and depletion properties, we then incorporated an electron-withdrawing phenyl sulde unit of 3,5bis(triuoromethyl)benzenethiol into the PS, which could more highly sensitive and selective to GSH than other related thiols. Within tumor cells, CyI-S-diCF 3 was specically activated by GSH, leading to the GSH decrease. The consumption of GSH not only elevated the intracellular ROS level, but also strengthened the 1 O 2 production efficiency under the NIR light irradiation, thus breaking the redox homeostasis in cancer cells. Furthermore, this disrupted redox homeostasis effectively induced mitochondrial membrane potential (MMP) dysfunction, followed by resulting in stronger and earlier apoptosis. Overall, we provide a new perspective on the design principle of organic photosensitizer to break the redox homeostasis of tumor cells.
The NMR spectra data were recorded on a Bruker Avance III 400 spectrometer, using TMS as the internal reference. High resolution mass spectra (HRMS) were performed using a LTQ Orbitrap XL spectrometer in the electrospray ionization (ESI) mode. Absorption and NIR uorescence emission spectra were obtained on an UV-3600 spectrophotometer (Shimadzu) and a Hitachi F4700 uorescence spectrometer, respectively. The cells imaging was conducted in an inverted uorescence microscope (Carl Zeiss, Axio Observer A1). TLC analysis was performed on silica gel plates and column chromatography was conducted over silica gel (mesh 200-300) columns, obtained from the Jiangyou Yantai Co. Ltd (Shandong, China).

Synthesis of CyI-S-diCF 3
To a dry 25 mL round bottom charged with CyI (30 mg, 0.033 mmol) dissolving in 3 mL anhydrous DMF, 3,5-bis(tri-uoromethyl)benzenethiol (9.5 mg, 0.038 mmol) was added rapidly, and then the mixed solution was stirred for 12 h at room temperature under N 2 atmosphere. Aer reaction nished, the mixture was evaporated in vacuum, and the crude product was puried by silica gel chromatography using CH 2 Cl 2 /CH 3 OH (15 : 1, v/v) as the eluent to obtain CyI-S-diCF 3 as a dark green solid (20 mg, 54%). 1

Fluorescence detection of GSH activity
CyI-S-diCF 3 was dissolved in PBS/DMSO buffer (10 mM, pH 7.4, 7/3, v/v, 37°C) with a concentration of 10 mM for the detection of GSH. With the addition of various concentrations of GSH (0-100 mM) to the CyI-S-diCF 3 solution, the uorescence intensity of the mixture at 823 nm was recorded aer incubation at 37°C for 5 min. (l ex = 780 nm). For time-dependent GSH response assay, upon GSH (100 mM) being added into the CyI-S-diCF 3 solution at 37°C, the FL spectra of the mixture at different time points (0-50 min) was measured in the range from 795 to 900 nm with the excitation of 780 nm. For the GSH inhibition experiment, CyI-S-diCF 3 (10 mM) solution was treated with NMM (100 mM), followed by the addition of GSH (100 mM). Aer that, the mixture was further incubated for 5 min at 37°C, and then the uorescence intensity of above solution was measured immediately.

Determination of singlet oxygen quantum yield (F D )
The singlet oxygen quantum yield (F D ) was tested by adjusted method according to literature. Typically, CyI-S-diCF 3 was dissolved in PBS/DMSO buffer (10 mM, pH 7.4, 7/3, v/v, 1% tween 80, 37°C) with 100 mM of GSH for 5 min, then the DPBF stock solution was added in above solution to adjust the absorbance at 418 nm was about 1.0. Aerward, UV-vis-NIR absorption spectra were recorded aer the mixed solution was exposed to 808 nm laser irradiation with a power density of 0.33 W cm −2 for various time. Indocyanine green (ICG) was tested under the same experimental procedures as the reference. At last, the singlet oxygen quantum yield was calculated by the following equation: where k (PSs) and k (ICG) were the decomposition rate constants of the absorbance at 418 nm of DPBF in the presence of the photosensitizer and ICG, respectively. F D represents the singlet oxygen quantum yield of the tested photosensitizer; F ICG represents the singlet oxygen quantum yield of ICG (F ICG = 0.2% in water); F is the correction factor which is calculated by the following equation: O.D. is the absorbance of the mixture at 808 nm.

Extracellular depletion of GSH
Briey, GSH (100 mM) was mixed with PBS or CyI-S-diCF 3 (10 mM) in PBS/DMSO buffer (10 mM, pH 7.4, 7/3, v/v). Aer being incubated at 37°C for 60 min, 200 mL of the above suspension and 1.0 mL of the DTNB solution (10 mM) were added into the working solution and further incubated for 5 min. Aerward, the absorbance at 412 nm was recorded via a UV-vis spectrophotometer.

Cell culture and in vitro cytotoxicity
Murine breast cancer cell (4T1) was obtained from the Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS with 5% CO 2 at 37°C. The cytotoxicity was measured using a standard CCK-8 assay. Typically, 4T1 cells were seed into 96-well microplates at an initial density of 3.5 × 10 3 cells/well. Aer 24 h of adherence and stable growth, the cells were incubated with RPMI-1640 medium containing different concentrations of CyI-S-diCF 3 for 4 h, then irradiated by 808 nm laser (1.0 W cm −2 ) for 5 min. As a control, a similar assay without light irradiation was performed. Aer further incubation for 2 h, CCK-8 assay was performed according to the protocol.

Dead/live cell co-staining
Firstly, 4T1 cells (3.5 × 10 3 ) were seeded into the 96-well plate and cultured for 24 h. Aer reaching 80% conuence, the cells were treated with CyI-S-diCF 3 (4.0 mM) at 37°C for 4 h and then irradiated by 808 nm laser (1.0 W cm −2 ) for 5 min. Cells without irradiation were utilized as controls. Aerwards, the cells were co-stained with calcein AM and PI for 30 min and imaged by the uorescence microscope, where live cells were stained in green and dead cells in red, respectively.

Intracellular ROS detection by DCFH-DA
Reactive Oxygen Species Assay Kit was used according to the manufacture instruction. Briey, 4T1 cells were rstly seeded into 96-well plates with the cell density of 3 × 10 3 cells per well and cultured for 24 h. Aerwards, the medium was replaced by fresh RPMI-1640 medium (control group, 808 nm laser group) or 4.0 mM of CyI-S-diCF 3 (CyI-S-diCF 3 group, CyI-S-diCF 3 + 808 nm group) and incubated for another 4 h, followed by treating with 10 mM DCFH-DA for 30 min at 37°C. Aer that, the cells were irradiated with 808 nm laser for 5 min at a power density of 1.0 W cm −2 . Fluorescence images were captured by inverted uorescence microscope.
2.10 Detection of mitochondrion membrane potential by JC-1 probe 3 × 10 3 4T1 cells were regularly seeded and cultured in 96-well plates for 24 h at 37°C. Then, cells were incubated with CyI-S-diCF 3 (4.0 mM) for 4 h and blank medium as negative control groups. Aer that, the cells in light groups were exposed to 808 nm laser (1.0 W cm −2 ) for 5 min, and then all the cells were cultured for another 2 h. Subsequently, the cells were stained by JC-1 kit according to the instruction manual. Ultimately, uorescence microscope was applied to determine the mitochondrion membrane potential.

Evaluation of intracellular GSH depletion in 4T1 cells
According to the method in the previous literature with some modications, 4T1 cells were seeded in 24-well plates at a density of 5 × 10 4 cells per well and cultured for 24 h. The cells were treated with PBS or CyI-S-diCF 3 (4.0 mM) for 4 h at 37°C. Subsequently, all the cells were digestive by trypsin-EDTA and centrifuged at 1000 rpm for 4 min. Then 80 mL of Triton-X-100 lysis buffer (0.4%) was used to lyse the cells on ice for 20 min. Aerward, the lysates were centrifuged at 6000 rpm for 5 min, and 50 mL of supernatant was mixed with DTNB (150 mL, 0.5 mM). Finally, the absorbance (412 nm) of the solutions was measured using a microplate reader.

Statistical analysis
All data were expressed in this study as mean result ± standard deviation (S.D.). The statistical analysis was performed using Matlab soware. The statistical differences between two groups were conducted by unpaired Student's two-sided t test analysis. Asterisks indicate signicant differences (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Synthesis and activatable property of CyI-S-diCF 3
CyI-S-diCF 3 was easily prepared through the sulydryl nucleophilic substitution reaction of chlorine atoms on iodinated heptamethine cyanine dye (CyI) by 3,5-bis(triuoromethyl) benzenethiol (BTBT) (Fig. S1 †). The intermediate of CyI was synthesized according to the previous ref. 41 The nal product was fully characterized by 1 H NMR, 13 C NMR, 19 F NMR and high-resolution mass spectrum (ESI-HRMS). Typically, CyI-S-diCF 3 generated 13 proton signals in the downeld region as shown in the Fig. S6 † from aromatic carbon and polymethine bridge, while the remaining 28 proton signals were found in the aliphatic region, indicating the accuracy of the structure. In addition, the coupling constant of polymethine bridge was calculated to be 16 Hz, which was larger than 15 Hz, suggested that CyI-S-diCF 3 was mainly existing in the E-geometry. Moreover, 13 C NMR and 19 F NMR spectra have furtherly conrmed that the structure possessed the CF 3 functional group (Fig. S7 and S8 †), and ESI-HRMS also showed an expected molecular weight (Fig. S9 †). Thus, those characterization results were consistent with the proposed structure. To investigate the sensitivity toward GSH, photochemical properties of CyI-S-diCF 3 was determined based on GSH concentration and response time in PBS/DMSO mixtures (10 mM, pH 7.4, 7/3, v/v). Upon the addition of GSH (0-100 eq.) to the CyI-S-diCF 3 solution (10 mM), the absorbance at 798 nm increased gradually due to the formation of a water-soluble product triggered by GSH (Fig. 1A). Meanwhile, the uorescence intensity of CyI-S-diCF 3 at 823 nm increased proportionally to the GSH concentration (Fig. 1B). This remarkable enhancement emission could be attributed to the restriction of the photoinduced electrontransfer (PET) effect from BTBT moiety to heptamethine. Of note, a good linear relationship was observed between the uorescence intensities of CyI-S-diCF 3 and the GSH concentration (Fig. S2 †). In addition, the time-dependent uorescence changes of CyI-S-diCF 3 with various concentration of GSH was also investigated, where the intensity variations rapidly increased and reached a plateau within 50 min (Fig. S3 and  S4 †). Although CyI-S-diCF 3 exhibited a similar sensitivity toward GSH compared with the reported NIR photosensitizer, it possessed a longer excitation wavelength (700-900 nm) than other one, suggesting its potential to precise photodynamic therapy. 42,43 Subsequently, the selectivity of CyI-S-diCF 3 was evaluated against similar biothiols such as L-cysteine (Cys) and L-homocysteine (Hcy). As shown in Fig. 1C, only GSH treated group showed a signicant change in uorescence intensity, while Cys and Hcy treated group could not exhibited obvious uorescence signal, thus suggesting a higher GSH-specic responsiveness of CyI-S-diCF 3 then some reported AIE based GSH activatable photosensitizers (e.g., TPEPY-S-Fc), as it could not able to distinct the GSH from other closely related thiols (e.g., Cys, Hcy). 44 It is worth noting that the activation efficiency of CyI-S-diCF 3 by GSH was signicantly decreased aer the addition of N-methylmaleimide (NMM, a GSH inhibitor) (Fig. 1D), further implying that CyI-S-diCF 3 was a GSH-specic activation probe.

Extracellular singlet oxygen ( 1 O 2 ) generation and GSH depletion
Aer conrming the excellent selectivity of CyI-S-diCF 3 to GSH, the 1 O 2 generation capability of CyI-S-diCF 3 in the absence and presence of GSH under 808 nm irradiation was then assessed by using 1,3-diphenylisobenzofuran (DPBF) as the 1 O 2 trapping agent. Compare to the DPBF group, the PBS/DMSO solution with DPBF and CyI-S-diCF 3 still showed a slightly reduction in DPBF absorbance at 418 nm under the 808 nm irradiation. Nevertheless, the absorption intensity of DPBF decreased rapidly with a continuous irradiation time from 0 to 180 s aer CyI-S-diCF 3 was pretreated with GSH at 37°C for 5 min, indicating a superior 1 O 2 production efficiency ( Fig. 2A). Therefore, it is reasonable to infer that GSH-activated CyI-S-diCF 3 could simultaneously favor radiative decay pathway and ISC transitions rate. The 1 O 2 generation efficiency was further quantied by calculating the singlet oxygen quantum yield (F D ) of CyI-S-diCF 3 before and aer GSH response through using ICG as the reference (F D = 0.2% in water). Signicantly, CyI-S-diCF 3 exhibited a higher quantum yield of 0.66% aer triggered by reaction with GSH, while the CyI-S-diCF 3 in absence of GSH resulted in the relative lower quantum yields of 0.45% (Fig. S5 †), indicating that CyI-S-diCF 3 could consume the GSH and followed by enhancing the 1 O 2 generation capability. Moreover, the 1 O 2 generation efficiency of CyI-S-diCF 3 treating with GSH displayed a signicant positive correlation with laser density (Fig. 2B), suggesting that laser power density could serve as an "accelerator" to promote the 1 O 2 production.
In light of the good GSH-specic responsiveness, the capacity of CyI-S-diCF 3 to deplete GSH was evaluated by using the 5,5 ′ -dithiobis-(2-nitrobenzoic acid) (DTNB), which could react with GSH to produce yellow derivative 5-thio-2nitrobenzoic acid (TNB). As illustrated in Fig. 2C, the absorbance peak of TNB at 412 nm decreased distinctly with the addition of CyI-S-diCF 3 , indicating that the GSH could be effectively consumed by CyI-S-diCF 3 . Taken all together, the GSH-activated CyI-S-diCF 3 with GSH depletion and enhanced 1 O 2 generation properties could be used as a ROS amplier to disrupt the redox homeostasis in microenvironment of cancer cells (Fig. 2D).

In vitro cytotoxicity evaluation
The anticancer effect of CyI-S-diCF 3 against 4T1 cells was investigated using a standard CCK-8 assay. Tumor cells were treated with various concentrations of CyI-S-diCF 3 in dark or under 808 nm light irradiation, and then the cell viability was measured. As shown in Fig. 3A, CyI-S-diCF 3 exhibited negligible toxicity to cells at the low concentrations in dark, whereas obvious cytotoxicity (below 80%) was noted at concentrations up to 4.0 mM, which was partially attributed to elevation of ROS levels in tumor cells cause by GSH consumption. In addition, the cytotoxicity of CyI-S-diCF 3 from the light group showed a signicant concentration dependence. The viability of 4T1 cell dropped to almost 10% when the cells pre-treated with CyI-S-diCF 3 (4.0 mM) were exposed to 808 nm irradiation (1.0 W cm −2 , 5 min), suggesting its great ROS generation efficiency.
Subsequently, live/dead cell staining assay was conducted to visualize the PDT efficacy of CyI-S-diCF 3 (Fig. 3B). Calcein acetoxymethyl ester (calcein-AM) was hydrolyzed into calcein and emitted green uorescence in living cells, whereas propidium iodide (PI) only exhibited red uorescence in dead cells duo to its poor cell membrane permeability. The cells treated with PBS and PBS with light showed a bright green uorescence, indicating light alone did not harm cells. In contrast, some red uorescence was observed in 4T1 cells incubated with CyI-S-diCF 3 in the dark, demonstrating slightly killing effect causing by the reduction of GSH content in cells. Moreover, cells cultured with CyI-S-diCF 3 under 808 nm irradiation (1.0 W cm −2 , 5 min) displayed intense red uorescence and negligible green uorescence, again illustrating the strong antitumor efficacy of CyI-S-diCF 3 . These results showed that CyI-S-diCF 3 can be used as a highly effective photosensitizer to induce tumor cell death triggered by intracellular GSH depletion and sufficient 1 O 2 production under light.

Intracellular ROS and GSH detection
Based on the good GSH specicity, CyI-S-diCF 3 could exhibited intense uorescence signal and elevate 1 O 2 generation efficiency aer activated by GSH, following disrupting the redox homeostasis in tumor cells. The resulting oxidative stress further promoted mitochondrial membrane potential damage, leading to cell apoptosis (Fig. 4A). Encouraged by the GSH depletion property of CyI-S-diCF 3 at extracellular level, the intracellular GSH content was rstly examined (Fig. 4B). The untreated control group showed a negligible change in GSH level, whereas the cells incubated with CyI-S-diCF 3 could induce approximately 22% reduction of GSH, suggesting that CyI-S-diCF 3 could promote the consumption of GSH in tumor cells.
Next, in order to assess the GSH accelerated photodynamic effect, the ROS generation efficiency of CyI-S-diCF 3 in 4T1 cells was evaluated using DCFH-DA as the detection probe. As shown in Fig. 4C, weak green uorescence was clearly observed in 4T1 cells with the treatment of CyI-S-diCF 3 in dark, indicating the down-regulation of GSH and the enhancement of ROS. Thus, compared to other GSH activatable NIR photosensitizers, CyI-S-diCF 3 exhibited the signicant GSH consumption property, implying that it could be used as an inducer to break the redox homeostasis in tumor cells. 43,45 Moreover, the cells treated with CyI-S-diCF 3 under 808 nm irradiation (1.0 W cm 2 , 5 min) exhibited intense green uorescence, implying the presence of large amounts of 1 O 2 . However, no green uorescence signal was detected in the PBS treated cells in dark or under light. These results rmly demonstrated that GSH and light irradiation could sequentially manipulate the ROS production efficiency of CyI-S-diCF 3 in tumor cells. The mitochondrial membrane potential (MMP) assay was then performed as it could be a signicant index to evaluate the extent of the mitochondrial destruction. From the Fig. 4D, we clearly found that the cells in control group displayed solely strong red uorescence because of its good integrity in mitochondria. However, both intensive green uorescence (JC-1 monomers) and red uorescence (JC-1 aggregates) were observed in cells treated with CyI-S-diCF 3 in dark, suggesting its partial potential loss to the mitochondrial membrane. Notably, under 808 nm light irradiation (1.0 W cm 2 , 5 min), the cells incubated with CyI-S-diCF 3 exhibited strong green uorescence but weak red uorescence, indicating a severely decrease in mitochondrial membrane potential. In short, CyI-S-diCF 3 could be served as a smart photosensitizer to effectively induce cells apoptosis causing by GSH down-regulation and enhancement of 1 O 2 generation. Data are presented as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (B) Calcein AM (green) and propidium iodide (red) co-staining fluorescence imaging of 4T1 cells after different treatments. 808 nm light irradiation (1.0 W cm 2 , 5 min) was conducted after cells were incubated with CyI-S-diCF 3 (4.0 mM) for 4 h. Scale bar: 100 mm.

Conclusion
In summary, we reported a new GSH activated photosensitizer based on a heptamethine cyanine scaffold. The single-atom replacement of hydrogen for iodine signicantly enhanced the photodynamic efficiency of heptamethine cyanine due to the heavy-atom effect (HAE). More importantly, the recognition units of 3,5-bis(triuoromethyl)benzenethiol on photosensitizer showed a higher sensitivity and selectivity toward GSH then other closely related biothiols. Aer triggering by cellular GSH, CyI-S-diCF 3 could switch to "on" state and produced abundant of toxic 1 O 2 under 808 nm light irradiation. This highlevel of ROS generation and antioxidant GSH elimination could effectively break the cellular redox homeostasis, leading to augmented oxidative stress. In vitro experiment demonstrated excellent GSH depletion-assisted enhanced PDT effects of the CyI-S-diCF 3 against tumor cells. Thus, this study may provide a new strategy for designing activatable NIR photosensitizer with disrupted redox homeostasis property.

Conflicts of interest
There are no conicts to declare.